Differentiation of Human Induced Pluripotent Stem Cells (iPSCs)–derived Mesenchymal Progenitors into Chondrocytes

Induced pluripotent stem cells (iPSCs) generated from human sources are valuable tools for studying skeletal development and diseases, as well as for potential use in regenerative medicine for skeletal tissues such as articular cartilage. To successfully differentiate human iPSCs into functional chondrocytes, it is essential to establish efficient and reproducible strategies that closely mimic the physiological chondrogenic differentiation process. Here, we describe a simple and efficient protocol for differentiation of human iPSCs into chondrocytes via generation of an intermediate population of mesenchymal progenitors. These methodologies include step-by-step procedures for mesenchymal derivation, induction of chondrogenic differentiation, and evaluation of the chondrogenic marker gene expression. In this protocol, we describe the detailed procedure for successful derivation of mesenchymal progenitor population from human iPSCs, which are then differentiated into chondrocytes using high-density culture conditions by stimulating with bone morphogenetic protein-2 (BMP-2) or transforming growth factor beta-3 (TGFβ-3). The differentiated iPSCs exhibit temporal expression of cartilage genes and accumulation of a cartilaginous extracellular matrix in vitro, indicating successful chondrogenic differentiation. These detailed methodologies help effective differentiation of human iPSCs into the chondrogenic lineage to obtain functional chondrocytes, which hold great promise for modeling skeletal development and disease, as well as for potential use in regenerative medicine for cell-based therapy for cartilage regeneration. Key features • Differentiation of human iPSCs into chondrocytes using 3D culture methods. • Uses mesenchymal progenitors as an intermediate for differentiation into chondrocytes.


Background
Human induced pluripotent stem cells (iPSCs) hold great potential for regenerative medicine, as they can be tailored to patient-specific cells for replacing musculoskeletal tissues with limited inherent repair capacity, such as articular cartilage.However, the pluripotent nature of human iPSCs poses a significant challenge in directing their differentiation towards specialized cell types, such as chondrocytes.Various in vitro strategies have been reported for inducing chondrogenic differentiation of human pluripotent stem cells (Guzzo and Drissi, 2015;Wu et al., 2021;Dicks et al., 2023;Khan et al., 2023;Lamandé et al., 2023;Zujur et al., 2023).Some of these approaches involve initial pre-differentiation steps within embryoid bodies, followed by dispersal and high-density culture to promote cell-cell interactions that mimic pre-cartilage condensation during skeletal development (Guzzo and Drissi, 2015;Dicks et al., 2023).Other studies have investigated the efficacy of stage-specific administration of developmentally relevant growth factors, such as bone morphogenetic proteins (BMPs) and transforming growth factors (TGFs), in controlling the induction of human pluripotent stem cells into the chondrogenic lineage and subsequent chondrocyte differentiation (Suchorska et al., 2017;Mahboudi et al., 2018).We have previously developed a method to induce chondrogenic differentiation from human iPSCs using mesenchymal-like progenitor cells as the intermediate population (Guzzo et al., 2013;Guzzo and Drissi, 2015).These progenitor-like cells exhibit molecular and functional properties similar to adult mesenchymal stem cells (MSCs) as a means to limit the developmental potency of iPSCs (Guzzo et al., 2013;Guzzo and Drissi, 2015;Khan et al., 2023).A readily expandable source of iPSCs-derived multipotent progenitors (iMSCs) with high chondrogenicity could potentially provide a vast supply of cells for regenerative medicine applications, as they can be used as an alternate source of adult MSCs, such as bone-marrow-derived MSCs and adipose-derived stem cells.These MSCs derived from iPSCs (referred here as iMSCs) have the potential to overcome clinical barriers for therapeutic applications of adult MSCs, as these iMSCs represent infinitive cell sources (ex vivo and in vivo) as well as isogenic MSCs providing renewable cell sources for MSCs.Here, we provide detailed step-by-step protocols for chondrogenic differentiation of human iPSCs from a naive, pluripotent state and via the derivation of a scalable, mesenchymal-like progenitor intermediate as we have described previously (Khan et al., 2023).We first describe the derivation of iMSCs from human iPSCs using direct plating and embryoid bodies formation.We then describe the differentiation of iMSCs into chondrocytes using 3D pellet culture and micromass culture method.We also describe the histological methods and gene expression analyses used to evaluate chondrogenic differentiation.The schematic representation of the step-by-step method used for differentiation of iPSCs to chondrocytes is shown in Figure 1.

Procedure
A. Thawing and recovering human iPSCs 1. Pre-warm an appropriate volume of mTeSR TM Plus medium to room temperature in a 50 mL Falcon tube (refer to Note 1). 2. Prepare the Geltrex-coated 6-well plates at least 1 h before iPSC thawing.3. Retrieve the cryogenic vial containing frozen iPSCs from the liquid nitrogen promptly and place it in a 37 °C water bath.4. Hold the cap of the cryogenic vial and gently swirl it to thaw the cells, ensuring that the cap remains above the water level.5. Once most cells are thawed and only a few ice crystals remain, remove the cryogenic vial from the water bath, spray it with 70% ethanol, and transfer it to a laminar hood.

6.
Carefully open the cap of the vial and transfer the cell suspension into a 15 mL Falcon tube containing the 10 mL pre-warmed Ham's F12 nutrient mix. 7. Rinse the cryogenic vial with 1 mL of F12 medium and add the solution to the same Falcon tube, gently mixing the cells by pipetting.8. Centrifuge the Falcon tube at 300× g for 5 min at room temperature and discard the supernatant.9. Gently resuspend the iPSC pellets in 2 mL of mTeSR TM Plus medium by gently pipetting up and down 3-4 times using 1 mL pipette tips, being cautious not to break the iPSC colonies into single-cell suspension.10.Add 2 μL of ROCK inhibitor (1,000× stock solution) to the cell suspension to enhance iPSC survival (refer to Note 3).11.Prepare a 6-well plate by removing the excess Geltrex from the pre-coated 6-well plate and then wash the wells with 2 mL of 1× DPBS.Aspirate the PBS and slowly transfer the iPSC suspension into the plate.Distribute one cryogenic vial of iPSCs into 1-2 wells of the 6-well plate.12. Gently tilt the culture plate back and forth and move it left and right several times to disperse cells evenly across the entire well (refer to Note 4).13.Incubate the plate in a 37 °C, 5% CO2 incubator and allow the cells to culture overnight.14.The following day, replace the medium with fresh mTeSR TM Plus medium, excluding the Rock inhibitor Y-27632.15.Subsequently, change media every day, replacing with fresh 2 mL of mTeSR TM Plus medium per well in the 6-well plate.Allow the iPSCs to grow for 4-5 days.Upon reaching 70%-80% confluence, the cells can be passaged (refer to Note 5).

B. Passage iPSCs using ReLeSR method
Typically, we pass iPSCs every 4-5 days, when they reach a confluence of 70%-80% and can be passaged as cell aggregates.For the passaging of iPSCs as cell aggregates, ReLeSR is routinely employed in our protocols (refer to Note 6).mTeSR TM is an enzyme-free reagent that selectively detaches undifferentiated cells and generates optimal-size aggregates.
1. Before initiating the protocol, ensure that new 6-well plates are coated with Geltrex at least 1 h in advance.Additionally, pre-warm the required volume of mTeSR TM Plus medium to room temperature.2. Carefully aspirate the spent medium from the wells containing cells and perform a single wash with 2 mL of DPBS without Ca 2+ and Mg 2+ .No selective removal of differentiated regions of iPSCs is necessary.3. Add 1 mL of ReLeSR into each well of the 6-well plate.Swirl the plate gently to ensure uniform distribution of ReLeSR over the entire cell surface.Within one minute, aspirate the ReLeSR solution from the wells.4. Add 1 mL of mTeSR TM Plus medium to each well of the 6-well plate and monitor cell dissociation under an inverted microscope. 5. Once iPSCs start to exhibit separation and rounding up at the periphery, they are ready to be removed from the wells using a cell lifter.6. Gently pipette the cell mixtures up and down 5-6 times to disperse large colonies into small clumps.
Collect all resulting cell aggregates into a 15 mL tube (refer to Note 8). 7. Centrifuge the 15 mL tube at 300× g for 5 min at room temperature and discard the supernatant.Add 2 mL of mTeSR TM Plus medium to the cell aggregate in 15 mL tube.8. Prepare the 6-well plate by removing any remaining Geltrex solution from the pre-coated plate.Wash the plate once with 2 mL of 1× PBS to remove any remaining Geltrex.9. Transfer the 0.5 mL of mTeSR TM Plus medium containing cell aggregate to each well of the 6-well plate and uniformly distribute the cell aggregate onto the plate surface.Add 1.5 mL of mTeSR TM Plus medium to each well to make a total volume of 2 mL.Generally, after 80% confluence of iPSC colonies, we pass the cells into 1:4 split ratio.10.Supplement the culture system with 2 μL of Rock inhibitor Y-27632 (1,000×, working concentration 10 μM) to enhance the survival of iPSCs.11.Gently tilt and move the plate in quick back and forth and side to side motions to evenly distribute the cell aggregates across the plate surface.Subsequently, return the plate to the 37 °C incubator (refer to Note 10).12. Daily, refresh the mTeSR TM Plus medium and observe the cultures under suitable conditions to monitor growth until the subsequent passaging or banking (refer to Note 11).

C. Derivation of mesenchymal progenitors (MSCs) from iPSCs using direct plating method
We have devised a streamlined and effective protocol for the feeder-free differentiation of iPSCs into MSCs in 1.Before initiating the differentiation process, coat each well of a 6-well plate with 1 mL of 0.1% gelatin solution and allow it to incubate at room temperature for 1 h.Subsequently, wash the coated plates once with 1× PBS. 2. Remove the mTeSR TM Plus medium from the routine iPSC cultures in 6-well plates and wash the cells with sterile PBS.Apply 1 mL of ReLeSR to each well and gently swirl the plate to ensure even coverage of the cell surface.Within a minute, aspirate the ReLeSR solution from the wells.3. Add 1 mL of mTeSR TM Plus medium to the wells and, using a cell lifter, remove the colonies from the plate.Gently pipette the cell mixtures up and down 3-4 times to break down large colonies into smaller clumps.Collect all resulting cell aggregates into a 15 mL tube.4. Centrifuge the 15 mL tube at 300× g for 5 min at room temperature and discard the supernatant.Wash the cell aggregates with 1× PBS and then add 1 mL of mesenchymal stem cell induction and growth medium (referred to as MSC growth medium) to each tube. 5. Plate the iPSCs onto gelatin-coated 6-well plates.Employ a cell culture split ratio of 1:1 or 1:2, where cells harvested from one well are distributed onto one or two new wells.This step marks the initial passage, referred to as iPS-MSCs or iMSCs passage 0 (p0).6. Change the medium every 2-3 days.After 7-10 days, the cultures will exhibit a mixture of flattened cuboidal and elongated spindle-shaped cells, indicating successful differentiation (refer to Note 12).Once a confluent monolayer is attained, proceed to the next passage.7. Apply 0.5 mL of 0.25% Trypsin-EDTA solution to each well of a 6-well dish and incubate at 37 °C for 3-5 min.Use repeated pipetting with a 1 mL pipettor to singularize the cells.Transfer the cell suspension to a 15 mL conical tube containing MSC growth medium.Centrifuge the cells at 300× g for 5 min and discard the supernatant.8. Resuspend the cell pellet in MSC growth medium and perform cell counting.Prepare a cell suspension at a concentration of 0.25 × 10 6 cells/mL.Plate 1 mL of the cell suspension into each well of a 6-well culture plate, which has been precoated with gelatin solution.This will mark the iPS cell-MSCs (iMSCs) passage 1. 9. Maintain the cells in a humidified atmosphere with 5% CO2 at 37 °C, with medium changes every 2-3 days.Within two weeks, the cells will adopt a fibroblastic, spindle-like morphology indicative of mesenchymal progenitor cells (Figure 2) (refer to Note 13).When cultures reach 90% confluence, split the cells using 0.25% Trypsin-EDTA solution and seed them at a density of 1 × 10 4 cells/cm 2 onto gelatincoated plates.10. Assay the expression of stem cell genes (e.g., Oct4, Nanog, alkaline phosphatase, Klf4) and gene markers associated with the mesenchymal lineage (e.g., Twist1, Col1a1) by quantitative RT-PCR, as described below in step G11.Notably, stem cell genes will be suppressed in the mesenchymal-like population, while
1. Prepare fresh 2× MSC freezing medium and keep it on ice until use.
2. Obtain a single cell suspension of MSCs using the Trypsin-EDTA method, following the procedures detailed in steps C7-C9.3. Gently dislodge the cell pellets through finger tapping and resuspend the cells in MSC growth medium to achieve a single-cell suspension.4. Using a hemocytometer, perform cell counting and adjust the cell density to 2 × 10 6 cells/mL.5. Add an equal volume of cold 2× MSC freezing medium to the MSC cell suspension, ensuring thorough mixing.6. Aliquot 1 mL of the cell mixture into each cryogenic vial.Each cryogenic vial should contain 1 × 10 6 cells (refer to Note 17). 7. Swiftly place the cryogenic vials into the freezing container and maintain them at -80 °C overnight.8.The following day, transfer the cryogenic vials containing cells to a -80 °C freezer or a liquid nitrogen tank for sustained long-term storage.

E. MSC surface antigens analysis by flow cytometry
The iPSC-derived mesenchymal stem cells (iPSC-MSCs or iMSCs) exhibit comparable surface antigen expression to human bone marrow-derived MSCs (BM-MSCs).For the analysis of surface antigens, antibodies targeting human CD34, CD45, CD73, CD90, and CD105 can be employed (refer to Antibodies section).Human bone marrow-derived MSCs serve as positive control cells, while the negative markers include CD34 -and CD45-, and the positive markers comprise CD73 + , CD90 + , and CD105 + .
Protocol for flow cytometric analysis of surface antigens 1. Prepare the MSC single-cell suspension as described in steps C7-C9.
2. Gently break the cell pellets through finger tapping and resuspend the cells using cold 1% FBS in DPBS without Ca 2+ and Mg 2+ .3. Count the total number of cells using a hemocytometer and adjust the cell density to 2 × 10 6 cells/mL.4. Transfer 2 × 10 5 cells (100 μL cell suspension) into 1.5 mL centrifuge tubes (refer to Note 18). 5. Add the appropriate volume of MSC antibodies (refer to Antibodies section) to each tube and gently mix them by pipetting.6. Incubate the cell samples for 30 min at room temperature in the dark.7.After the incubation, wash the cells once with 1% FBS in DPBS without Ca 2+ and Mg 2+ .Finally, resuspend the cell samples in 250 μL of 4% formaldehyde-DPBS without Ca 2+ and Mg 2+ .8. Analyze the cell samples using a flow cytometer (refer to Note 20) to evaluate surface antigen expression as described in our previous paper (Khan et al., 2023).

F. Differentiation of iPSC-MSCs (iMSCs) into chondrocytes
The iPSC-MSCs (iMSCs) demonstrate a versatile capacity for multipotent differentiation encompassing osteogenesis, chondrogenesis, and adipogenesis.In this study, we explored two distinct in vitro methods for differentiating iMSCs into chondrocytes, as described below.
Micromass method for differentiation of iMSCs into chondrocytes 1. Expand human iMSCs in a 10 cm culture dish in MSC growth medium at 37 °C and 5% CO2 until they reach 75%-80% confluence.2. Aspirate culture medium and wash the cells once with 10 mL of PBS.Subsequently, apply 1 mL of 0.25% Trypsin-EDTA solution to the culture dish (refer to Note 21).Return the dish to the 37 °C incubator and incubate for approximately 3-5 min.Upon detachment of the cells, pipette gently and repeatedly using a 1 mL pipettor to disperse the iMSCs into single cells.3. Transfer the cell suspension to a 15 mL sterile conical polypropylene tube containing MSC growth medium.
Centrifuge the cells at 300× g for 5 min and discard the supernatant.4. Resuspend the cell pellet in 5 mL of MSC growth medium and perform gentle pipetting using a 1 mL pipettor to make single-cell suspension.If required, pass the cell suspension through a 40 μm nylon cell strainer.5. Cell counting is done using the Trypan Blue exclusion method with a hemocytometer and Trypan Blue solution or by employing an automated cell counter.Cells are subsequently diluted in MSC growth medium to achieve a final concentration of 25 × 10 6 cells/mL.6.For seeding, apply 10 μL drops of the diluted cell suspension onto 6-well culture plates (see Note 22).Add up to three high-density cell spots per well of a 6-well dish, ensuring adequate spacing between the drops (Figure 3A) These micromasses represent structural condensation of iMSCs during chondrogenic differentiation.Figure 3A shows the formation of three micromasses, which are equally spaced in the 6well plate (see Note 23). 7. Allow the cells to attach for 2 h at 37 °C in a humidified 5% CO2 incubator.Following attachment, carefully apply 1.5 mL of MSC growth medium containing 10 μM of ROCK inhibitor Y-27632 to each well.Return the plate to the cell culture incubator and maintain overnight.8.After 24 h, aspirate the medium and add 2 mL of chondrogenic differentiation medium to each well.
Continue incubation at 37 °C in a humidified 5% CO2 incubator.9. On day 2, add fresh chondrogenic differentiation medium containing BMP-2 or TGFβ3 to each well.
Replace the growth factor-supplemented medium every other day.Pellet formation method 1.To initiate chondrogenic differentiation using the pellet formation method, harvest the iPSC-derived MSCs (iMSCs) using 0.25% trypsin-EDTA.Following centrifugation of the cells at 100× g, resuspend them in MSC growth medium.2. Perform viable cell counting via microscopy on a hemocytometer or other cell counting equipment using Trypan Blue solution in a 1:2 dilution of the cell suspension from step 1. Aliquot 2.5 × 10 5 viable iMSCs in 0.5 mL of MSC growth medium into 15 mL conical tubes for pellet cultures.Centrifuge the cells at 100× g for 1 min and incubate them in the 15 mL conical tubes overnight at 37 °C and 5% CO2 with loosened caps to facilitate gas exchange.3. Within 24-48 h of pellet formation, verify that the cells have formed a spherical pellet at the bottom of each 15 mL tube (Figure 4). Figure 4 indicates the condensation of cells in the form of 3D-pellet culture at the bottom of 15 mL conical tube.If cells do not form a pellet under these conditions, coat a 15 mL conical tube with anti-adherence rinsing solution for 1 h, wash with PBS, and then add cells to allow pellet formation.

Figure 4. Chondrogenic differentiation of induced pluripotent stem cells (iMSCs) using pellet method
4. Aspirate the medium and replace it with 0.5 mL of chondrogenic differentiation medium supplemented with either BMP-2 or TGFβ3. 5. Subsequently, culture the pellets in chondrogenic medium with BMP-2/TGFβ3 for 7, 14, and 21 days, with careful medium exchange in each 15 mL conical tube using 0.5 mL of fresh chondrogenic medium containing BMP-2 or TGFβ3 every other day.

G. Histological assessment of chondrogenic matrix production
1. Harvest the cell pellets and wash them once in PBS.Subsequently, fix the pellets in formalin solution for a duration of 15 min.2. Perform two washes of the fixed pellets in PBS, followed by sequential dehydration steps in 1.5 mL microcentrifuge tubes using 50%, 70%, 95%, and 100% ethanol and then ethanol/xylene solution and 100% xylene.Each dehydration step should take 5 min (two repetitions, see Note 24).Embed the dehydrated pellets in low-melt paraffin.3. Use a microtome to section the paraffin-embedded pellets, creating sections of 5-7 μm thickness, and mount them onto Superfrost Plus slides.
Published: Nov 05, 2023 4. De-paraffinize and rehydrate the sections on slides by subjecting them to sequential washes in 100% xylene (two repetitions, each for 10 min), 100%, 95%, 70%, and 50% ethanol (two repetitions, each for 2 min), and finally, distilled water (for 5 min). 5. To detect sulfated proteoglycan deposits indicative of functional chondrocytes, rinse the formalin-fixed sections of paraffin-embedded pellets in an acetic acid solution for 3 min.Subsequently, stain the sections with Alcian Blue solution at room temperature for 30 min.After a brief rinse in acetic acid solution and running tap water for 5-10 min, counterstain the cell nuclei with Nuclear Fast Red Kernechtrot solution at room temperature for 5 min, followed by a rinse in running tap water until clear (Figure 3B).Alcian blue staining as shown in Figure 3B shows accumulation of proteoglycans, which demonstrate the deposition of extracellular matrix in micromass culture of iMSCs.A high-magnification image demonstrates cellular compaction and condensation indicating the development of cartilage/chondrocytes (Figure 3C).6.For visualization of acidic proteoglycan in cartilage tissues, stain the hydrated formalin-fixed sections of paraffin-embedded pellets with Safranin O solution at room temperature for 5 min.Remove excess stain by rinsing the sections under running tap water for 5-10 min.7. Dehydrate the slides in 70%, 95%, and 100% ethanol, then 100% xylene (two repetitions, each for 2 min).
Finally, mount coverslips using CytoSeal mounting medium to facilitate microscopic assessment of proteoglycan content, with Alcian Blue staining resulting in diffuse blue dye deposits, and Safranin O staining leading to diffuse orange-red deposits.

H. Quantitative PCR analyses of cartilage genes
For PCR analysis, RNA is extracted from cell pellets collected throughout the chondrogenic differentiation time course (refer to Note 13).13.Present the data as mean ± S.E.M. of at least three independent samples.Perform statistical comparisons between untreated and growth factor-treated groups using a two-tailed Student's t-test.Significance is assigned to P values < 0.05 (see Note 14).

9 Published:
Cite as: Khan, N. M. et al. (2023).Differentiation of Human Induced Pluripotent Stem Cells (iPSCs)-derived Mesenchymal Progenitors into Chondrocytes.Bio-protocol 13(21): e4874.DOI: 10.21769/BioProtoc.4874.Nov 05, 2023 mesenchymal markers will be significantly induced.11.For routine expansion, seed the cells at a density of 1 × 10 4 cells/cm 2 and maintain them in MSC growth medium (see Note 14).Keep track of the passage numbers.Establish a cell bank by freezing batches at each passage.At harvest, resuspend singularized cells in 1× cryopreservation medium and aliquot 1 × 10 6 cells per vial.Freeze the vials at -80 °C and subsequently transfer them to liquid nitrogen for long-term storage (see Note 15).

Figure 3 .
Figure 3. 3D micromass culture for chondrogenic differentiation of induced pluripotent stem derived mesenchymal progenitor cells (iMSCs).(A) Chondrogenic differentiation of iMSCs using the micromass method (B) and Alcian blue staining for the assessment of proteoglycan deposition.Alcian blue staining showed accumulation of proteoglycans, indicating deposition of extracellular matrix in AC-iMSC micromass culture.(C) High magnification image demonstrating cellular compaction and condensation indicating the formation of chondrocytes.Scale bar, 100 μm.

13 Published: Nov 05, 2023 Figure 5 .
Figure 5. Gene expression analyses by qPCR showing expression of mesenchymal genes COL1A1 and RUNX1 in the AC-iMSCs relative to their parental induced pluripotent stem cells (iPSCs).β-Actin served as the housekeeping gene and internal control.Data represented as fold change relative to respective parental iPSCs.